1、BS EN ISO11702:2009ICS 67.200.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal andvegetable fats andoils Enzymaticdetermination of totalsterols content (ISO11702:2009)Copyright European Committee for Standardization Provided by IHS under license with CEN
2、Not for ResaleNo reproduction or networking permitted without license from IHS-,-,-This British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31 January2010 BSI 2010ISBN 978 0 580 64650 8Amendments/corrigenda issued since publicationDate CommentsBS EN ISO 1
3、1702:2009National forewordThis British Standard is the UK implementation of EN ISO 11702:2009.The UK participation in its preparation was entrusted to TechnicalCommittee AW/307, Oil seeds, animal and vegetable fats and oils andtheir by products.A list of organizations represented on this committee c
4、an be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.Copyright European Committee for Standar
5、dization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11702 December 2009 ICS 67.200.10 English Version Animal and vegetable fats and oils - Enzymatic determination of
6、 total sterols content (ISO 11702:2009) Corps gras dorigines animale et vgtale - Dtermination enzymatique de la teneur en strols totaux (ISO 11702:2009) Tierische und pflanzliche Fette und le - Enzymatische Bestimmung des Gesamtsterin-Gehaltes (ISO 11702:2009)This European Standard was approved by C
7、EN on 18 November 2009. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standa
8、rds may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to
9、 the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Ne
10、therlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitati
11、on in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 11702:2009: ECopyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS EN ISO 11702:2009EN I
12、SO 11702:2009 (E) 3 Foreword This document (EN ISO 11702:2009) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products - Methods of sampling and analysis”, the secretar
13、iat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by June 2010, and conflicting national standards shall be withdrawn at the latest by June 2010. Attention is drawn to th
14、e possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following count
15、ries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spai
16、n, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 11702:2009 has been approved by CEN as a EN ISO 11702:2009 without any modification. Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking pe
17、rmitted without license from IHS-,-,-BS EN ISO 11702:2009ISO 11702:2009(E) ISO 2009 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is no
18、rmally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in
19、 the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepa
20、re International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that so
21、me of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 11702 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegatable fats and oils. Copyright European
22、 Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS EN ISO 11702:2009Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networki
23、ng permitted without license from IHS-,-,-BS EN ISO 11702:2009INTERNATIONAL STANDARD ISO 11702:2009(E) ISO 2009 All rights reserved 1Animal and vegetable fats and oils Enzymatic determination of total sterols content 1 Scope This International Standard specifies a method for the quantitative determi
24、nation of the total sterols content by means of an enzymatic staining test. The method is applicable to free and esterified sterols in animal and vegetable fats and oils, fatty foods and related products. The determination is applicable to sample quantities of 1 g to 2 g of fat. The method is not ap
25、plicable to dark coloured fats and oils, e.g. crude palm oil. The enzyme is not specific for cholesterol, but also oxidizes other 3-hydroxysterols. The method has not been tested for products fortified with sterols at higher levels. NOTE The method is technically equivalent to IUPAC method 2.4048and
26、 DGF standard method F-III 2 (91)7. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments)
27、 applies. ISO 661, Animal and vegetable fats and oils Preparation of test sample ISO 3696, Water for analytical laboratory use Specification and test methods 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 total sterols content wsterolsmass f
28、raction of sterols determined by the method specified in this International Standard NOTE 1 For vegetable fats and oils, the sterols content is expressed as -sitosterol; for animal fats, as cholesterol. NOTE 2 The total sterols content is expressed in milligrams per 100 g of fat. 4 Principle The tes
29、t sample is saponified and the sterols in the unsaponifiable matter are determined enzymatically. They are oxidized by cholesterol oxidase to cholestenone. The equimolar amount of hydrogen peroxide produced in the process oxidizes in the presence of catalase methanol to formaldehyde. In the presence
30、 of ammonium ions, with acetylacetone, it forms a yellow lutidine dye (3,5-diacetyl-1,4-dihydrolutidine). The latter is determined spectrophotometrically in the visible range at 405 nm. The concentration of the dye is equivalent to the amount of sterols. NOTE Cholesterol oxidase oxidizes cholesterol
31、 and other sterols having a hydroxy group in the 3-position. Therefore, phytosterols like stigmasterol and sitosterol are also determined. Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduction or networking permitted without license from
32、 IHS-,-,-BS EN ISO 11702:2009ISO 11702:2009(E) 2 ISO 2009 All rights reserved5 Reagents WARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational and personal safety measures shall be followed. Unless otherwise stated, use only reagen
33、ts of recognized analytical grade. 5.1 Water, complying with ISO 3696, grade 3 or better. 5.2 Isopropanol. 5.3 Acetone. 5.4 Acetyl acetone. 5.5 Suspension of cholesterol oxidase1)(EC 1.1.3.6) from Nocardia erythropolis, 15 U/ml. 5.6 Suspension of catalase (hydrogen peroxide oxido-reductase)1)(EC 1.1
34、1.1.6) from bovine liver. 5.7 Hydrochloric acid, c(HCl) = 8 mol/l. 5.8 Methanolic potassium hydroxide solution, c(KOH) = 0,5 mol/l. Dissolve 2,8 g potassium hydroxide in a small amount of hot methanol, cool, and dilute with methanol to 100 ml. 5.9 Ammonium phosphate buffer solution, adjusted to pH 7
35、. 5.10 Solution 1. Add 19,1 ml acetone (5.3) and 230 000 U catalase (5.6) to 50 ml buffer solution (5.9) in a 100 ml one-mark volumetric flask (6.4), and make up to the mark with water (5.1). 5.11 Solution 2. Add 0,26 ml acetylacetone (5.4) and 1,10 ml acetone (5.3) to 25 ml water (5.1) in a 50 ml o
36、ne-mark volumetric flask (6.4), and make up to the mark with water. 5.12 Solution 3. Prior to use, mix 3 volumes of solution 1 (5.10) with 2 volumes of solution 2 (5.11). NOTE Solution 3 can be kept in amber bottles for 3 months at 4 C provided it is prepared under sterile conditions. 6 Apparatus 6.
37、1 Test tubes, of diameter 18 mm. 6.2 Filter funnel. 6.3 Fluted filter suitable for the filter funnel (6.2). 1) A suitable ready-made test kid for the colorimetric determination of cholesterol in foodstuffs and other materials is available from R-Biopharm. This information is given for the convenienc
38、e of users of this International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results. Copyright European Committee for Standardization Provided by IHS under license with CENNot for ResaleNo reproduct
39、ion or networking permitted without license from IHS-,-,-BS EN ISO 11702:2009ISO 11702:2009(E) ISO 2009 All rights reserved 36.4 One-mark volumetric flasks, of capacities 25 ml, 50 ml, and 100 ml, ISO 10422class A. 6.5 Enzyme pipettes, of capacities 0,02 ml, ISO 75506, to 1 ml, ISO 6481class A. 6.6
40、Pipette, of capacity 5 ml, ISO 6481 class A. 6.7 Round bottomed flask, standard ground joint, of capacity 50 ml. 6.8 Test tubes with ground stoppers. 6.9 Spectrophotometer, set to 405 nm. 6.10 Glass cuvettes, pathlength 1 cm, suitable for the spectrophotometer (6.9). 6.11 Water bath, thermostaticall
41、y controlled at 37 C to 40 C. 6.12 Refrigerator, capable of maintaining a temperature of 4 C. 6.13 Glass beads. 6.14 Reflux condenser, standard ground joint. 7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or sto
42、rage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 55553. 8 Preparation of the test sample Prepare the test sample in accordance with ISO 661. Specific treatment of the test sample (filtration, melting, etc.) shall be ment
43、ioned in the test report. 9 Procedure 9.1 Saponification 9.1.1 Weigh 1 g to 2 g of the sample accurately to within 0,001 g into a 50 ml round bottomed flask (6.7). The sterol concentration in the test solution shall be between 0,02 g/l and 0,4 g/l. This requirement shall be taken into account during
44、 the weighing and diluting steps. In the case of saturated fats, the amount weighed shall be reduced, as otherwise free fatty acids formed after saponification and acidification are not completely removed during filtration and affect the determination. Ensure at all times that the solution obtained
45、is clear. 9.1.2 Add 10 ml of methanolic potassium hydroxide solution (5.8) and some glass beads (6.13). Heat the mixture and, when boiling, reflux for 25 min. 9.1.3 Transfer the still warm soap solution quantitatively into a 25 ml one-mark volumetric flask (6.4) and wash out the round bottomed flask
46、 with a few millilitres of isopropanol (5.2). 9.1.4 Pipette (6.5) 1 ml of hydrochloric acid (5.7) into the 25 ml one-mark volumetric flask, make up to the mark with isopropanol (5.2) and shake vigorously. Ensure at all times that the solution obtained is clear. 9.1.5 Place the flask with the mixture
47、 (9.1.4) in the refrigerator (6.12) and maintain it at 4 C for 20 min. 9.1.6 Next, filter the (turbid) solution as rapidly as possible through a fluted filter (6.3) and immediately use the filtrate for the enzymatic determination. Copyright European Committee for Standardization Provided by IHS unde
48、r license with CENNot for ResaleNo reproduction or networking permitted without license from IHS-,-,-BS EN ISO 11702:2009ISO 11702:2009(E) 4 ISO 2009 All rights reserved9.2 Enzymatic determination of the sterols content 9.2.1 Pipette (6.6) 5 ml of solution 3 (5.12) into a test tube (6.1) and add 0,4 ml of the filtrate (9.1.6). Mix thoroughly. 9.2.2 Transfer 2,5 ml of this mixture into a stoppered test tube (6.8) and add by pipette (6.5) 0,02 ml of the cholesterol oxidase suspension (5.5). Mix thoroughly. 9.2.3 Transfer the rest of the solution from 9.2.1 into anothe
